Carbonic anhydrase (CA), also called carbonate dehydratase, catalyzes the hydration of carbon dioxide in the reaction H.sub.2 O+CO.sub.2 .revreaction.HCO.sub.3.sup.- +H.sup.+. It accelerate, this reaction by a factor of over 10.sup.6 by virtue of a zinc ion located in a deep cleft about 15 .ANG. below the protein's surface and coordinated to the imidazole groups of three His residues. Water bound to the zinc ion is rapidly converted to HCO.sub.3.sup.-.
CA is crucial to the maintenance of pH in body fluids. A large quantity of acids enter the body, from both dietary and metabolic sources and consume intracellular and extracellular buffers. HCO.sub.3.sup.- is the most important buffer in the intracellular compartment. CA is needed to regenerate and reclaim HCO.sub.3.sup.-. In the kidneys, the reabsorption of HCO.sub.3.sup.- in the renal proximal tubule occurs via CA, which combines CO.sub.2 with the OH.sup.- ion that results from the splitting of water. The resulting HCO.sub.3.sup.- moves across the peritubular cell membrane to enter the extracellular HCO.sub.3.sup.- pool. The H.sup.+ secreted into the tubule lumen combines with a filtered HCO.sub.3.sup.-, forming H.sub.2 CO.sub.3, which is later dehydrated to form CO.sub.2 that diffuses into peritubular blood, leaving a reclaimed HCO.sub.3.sup.- ion. So essential is the maintenance of buffering capacity that in chronic renal failure, bone tissue may be used as a source of HCO.sub.3.sup.- to replace that lost in the urine. In red blood cells, CA speeds the reaction of water with carbon dioxide from tissues so that large amounts of CO.sub.2 are taken up before the blood leaves the capillaries. CA may be involved in the regulation of vascular tonus, since CA activity is inhibited by vasodilating drugs such as nitroglycerin in parallel with their vasodilating effect (Puscas, I. et al. (1997) Am. J. Hypertens. 10(1):124-128). Carbonic anhydrase is one of the key enzymes responsible for the secretion of cerebrospinal fluid. This secretion increases dramatically during postnatal life in mammals. The expression of carbonic anhydrase is developmentally regulated in several cells, such as erythrocytes and striated muscle fibers (Catala, M. (1997) Childs Nerv. Syst. 13(7):364-368). CA in the extracellular boundary layer of sarcolemma facilitates CO.sub.2 transport via the catalyzed hydration of CO.sub.2, thus maintaining the PCO.sub.2 gradient across the sarcolemma, and H.sup.+ released in that reaction protonate excreted NH.sub.3.sup.- which helps maintain the PNH.sub.3 gradient (Henry, R. P. et al. (1997) Am. J. Physiol. 262(6/2):R1754-R1761). In the placenta, carbonic anhydrase may provide ions for exchange with Na.sup.+, K.sup.+, and Cl.sup.- in transepithelial movement of ions and fluid, as well as facilitating CO.sub.2 diffusion. It can also be active in intermediary metabolism, such as glucorieogenesis, urea, and fatty acid synthesis (Ridderstrale, Y. (1997) Microsc. Res. Tech. 38(1-2):115-124).
Kinetic studies of CA folding have indicated the occurrence of con formational intermediates. Human CAI contains a cysteine residue, Cys212, which is unavailable for alkylation in the native state, but can be specifically modified with iodoacetate in the unfolded state. Bergenhem et al. concluded that the Cys-containing beta strand is part of a nucleation center that forms during the folding process. The beta strand is also partly involved in forming the bottom of the active site cavity (Bergenhem, N. et al. (1989) Int. J. Pept. Prot. Res. 33(2):140-145). Within the active site Leu198, Thr199 and His200 have been identified as important for binding of sulfonamide inhibitors which interact with the zinc ion (Chakravarty, S. et al. (1994) J. Mol. Biol. 243(2):298-309). Spectroscopic evidence has indicated that histamine, a CA activator, also binds to the entrance of the active site but not to the metal ion (Briganti, F. et al. (1997) Biochemistry 36(34):10384-10392).
Eight enzymatic and evolutionarily related forms of carbonic anhydrase are currently known to exist in humans: three cytosolic isozymes (CAI, CAII, and CAIII, two membrane-bound forms (CAIV and CAVII), a mitochondrial form (CAV), a secreted salivary form (CAVI) and a yet uncharacterized isozyme. Isoforms show a characteristic motif. (See, e.g., http//expasy.hcuge.ch). Though the isoenzymes CAI, CAII, and bovine CAIII have similar secondary structure and polypeptide-chain fold, CAI has 6 tryptophans, CAII has 7 and CAIII has 8 (Boren, K. et al. (1996) Protein Sci. 5(12):2479-2484). CAII is the predominant CA isoenzyme in the brain of mammals.
Inhibition and activation of CA provide information about CA stricture and activity. Vasodilating prostaglandins E1, E2 and I2 inhibit CA in vitro and in vivo and may inhibit the involvement of CA in gastric acid secretion. Nonsteroidal anti-inflammattory drugs which reduce the activity of cyclooxygenase and prostaglandin production have also been observed to activate CAI and CAII in a dose-dependent noncompetitive manner. The pre-prostaglandin cyclooxygenase appears to maintain an inverse relationship with CA, probably mediated by the pH variations associated with carbonic anhydrase activity (Puscas, I. (1996) J. Pharmacol. Exp. Ther. 277(3):1464-1466). Both prostaglandins E2 and I2 inhibit gastric acid output. Prostaglandin E2 inhibits egress of norepinephrine from sympathetic nerve terminals.
Histamine is a CA activator. Histamine is released in essentially every tissue of the body when it becomes damaged, inflamed, or is subject to allergic reaction. Histamine stimulates gastric acid secretion, increases smooth muscle contraction, and has a powerful vasodilator effect, to the extent that the increased capillary porosity it causes may lead to edema. The prostaglandins, which are equally ubiquitous and important in inflammation processes, include vasodilating and some vasoconstricting agents, and function in circulatory control by their action on the smooth muscle of the vessel wall (Isselbacher, K. J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York, N.Y.).
A number of disease states are marked by variations in CA activity The concentration of CAII in the cerebrospinal fluid (CSF) appears to mark disease activity in patients with brain damage. High CA concentrations have been observed in patients with brain infarction. Patients with transient ischaemic attack, multiple sclerosis, or epilepsy usually have CAII concentrations in the normal range, but higher CAII levels have been observed in the CSF of those with central nervous system infection, demertia, or trigeminal neuralgia (Parkkila, A. K. et al. (1997) Eur. J. Clin. Invest. 27(5):392-397). Colonic adenomas and adenocarcinomas have been observed to fail to stain for CA, whereas non-neoplastic controls showed CAI and CAII in the cytoplasm of the columnar cells lining the upper half of colonic crypts. The neoplasms show staining patterns similar to less mature cells lining the base of normal crypts (Gramlich T. L. et al. (1990) Arch. Pathol. Lab. Med. 114(4):415-419). Deficiency of CAII has been identified as the primary defect in osteopetrosis, a rare metabolic bone disease characterized by increase in skeletal mass due to a defect in development or function of the osteoclasts (Felix, R. et al. (1996) Eur. J. Endocrinol. 134(2):143-156).
Therapeutic interventions in a number of diseases involve altering CA activity. Ophthalmic disorders are commonly treated with carbonic anhydrase inhibitors such as acetazolamide. Topical preparations such as eye drops have been suggested as an additional and safer treatment for patients with uncontrolled medical glaucoma (Centofanti, M. (1997) Pharmacol. Res. 35(5): 481-485). Carbonic anhydrase inhibitors are also used to treat chronic renal failure (Suki, W. N. (1997) Kidney Int. Suppl. 59:S33-S35), Parkinson's Disease and tardive dyskinesia (Cowen, M. A. et al. (1997) J. Clin. Pharmacol. 17(3):190-193) epileptic seizures uncontrolled by other marketed agents (Reiss, W. G. (1 996) Ann. Pharmacother. 30(5):514-519). Adverse effects of acetazolamide treatment include kidney stones, metabolic acidosis, lethargy, appetite suppression, paresthesias, and rare blood dyscrasias.
CA activity can be particularly useful as an indicator of long-term disease condition, since the enzyme reacts relatively slowly to physiological changes. CAI and zinc concentrations have been observed to decrease in hyperthyroid Graves' disease (Yoshida, K. (1996) Tohoku J Exp Med 178(4):345-356) and glycosylated CAI is observed in diabetes mellitus (Kondo, T. et al. (1987) Clin. Chim. Acta 166(2-3):227-236). A positive correlation has been observed between CAI and CAII reactivity and endometriosis (Brinton, D. A. et al. (1996) Ann. Clin. Lab. Sci. 26(5):409-420; D'Cruz, O. J. et al. (1996) Fertil. Steril. 66(4):547-556).
The discovery of a new carbonic anhydrase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention or treatment of circulatory and neuronal diseases, inflammatior, and cancer.